Cardiac ischemia-reperfusion (IR) injury is the pathologic process underlying heart-attack (acute myocardial infarction, AMI). Despite decades of research, cardioprotective therapies are scant, and AMI kills >220,000 people annually in the US. In addition, >380,000 patients/yr. undergo predictable ischemia during cardiac surgery. The overall goal of this ongoing program is to develop new cardioprotective therapies for IR injury. This is a competing renewal application, and in the previous funding cycle we published 19 primary research articles directly attributable to this award (and 17 additional papers). During this time we identified two key signaling players in the endogenous cardioprotective phenomenon of acute ischemic preconditioning (IPC). First, we showed NO derived nitro-lipids is generated in IPC, and covalently adducts mitochondrial adenine nucleotide translocase 1 (ANT1). This causes mild uncoupling of mitochondrial oxidative phosphorylation, which is independently known to protect against IR injury. Second, we showed that the lysine deacetylase SIRT1 is necessary for acute cardioprotective signaling, and that SIRT1 transgenic mice are endogenously protected against IR injury. While these mechanisms may initially appear distinct, our preliminary data show that nitro-lipids covalently modify SIRT1, suggesting cross-talk between nitro-lipid & SIRT1 signaling. The downstream mechanisms which mediate cardioprotection by nitro-lipids and SIRT1 are poorly defined, but our preliminary data suggest the importance of alterations in metabolism and the stimulation of mitophagy, both of which are implicated in cardioprotection. Furthermore, we have developed a cell-based screening platform applicable to adult primary cardiomycytes, enabling discovery of small molecules which can engage these mechanisms to protect the heart from IR injury in-vivo. Overall, these observations have led to the central hypothesis that nitro-lipids & SIRT1 act in concert to protect the heart from IR injury via alterations in metabolism and mitophagy. In this project, we will exploit these endogenous protective pathways for therapeutic benefit, by delivering small molecules that modulate them both prior to ischemia AND at reperfusion. The end goal is to deliver novel cardioprotective molecules for pre-clinical development. To achieve this goal and test our hypothesis, we will pursue three following specific aims: Aim 1 will identify cardioprotective targets of nitro-lipid action, with a focus on ANT1 and mitophagy. Aim 2 will investigate cardioprotective targets of SIRT1, with a focus on metabolism. Aim 3 will develop screening hits, with a focus on molecules that impact mitophagy and metabolism. Addressing these aims will yield a series of small molecule candidates for pre-clinical development as cardioprotective therapeutics, and will enhance our understanding of the underlying mechanisms of cardioprotection.